an emerging tick-borne disease of humans is...

Pathogens 2013, 2, 544-555; doi:10.3390/pathogens2030544

pathogens

ISSN 2076-0817

www.mdpi.com/journal/pathogens

Article

An Emerging Tick-Borne Disease of Humans Is Caused by a

Subset of Strains with Conserved Genome Structure

Anthony F. Barbet 1,2,†,

*, Basima Al-Khedery 1,†

, Snorre Stuen 3, Erik G. Granquist

4,

Roderick F. Felsheim 5 and Ulrike G. Munderloh

5

1 Department of Infectious Diseases and Pathology, University of Florida, Gainesville, FL 32611,

USA; E-Mail: [email protected] 2 Emerging Pathogens Institute, University of Florida, Gainesville, FL 32611, USA

3 Department of Production Animal Clinical Sciences, Norwegian School of Veterinary Science,

Sandnes N-4325, Norway; E-Mail: [email protected] 4 Department of Production Animal Clinical Sciences, Norwegian School of Veterinary Science,

Oslo N-0033, Norway; E-Mail: [email protected] 5 Department of Entomology, University of Minnesota, St. Paul, MN 55108, USA;

E-Mails: [email protected] (R.F.F.); [email protected] (U.G.M.)

† These authors contributed equally to this work.

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +1-352-294-4119; Fax: +1-352-392-9704.

Received: 17 July 2013; in revised form: 29 August 2013 / Accepted: 2 September 2013 /

Published: 10 September 2013

Abstract: The prevalence of tick-borne diseases is increasing worldwide. One such emerging

disease is human anaplasmosis. The causative organism, Anaplasma phagocytophilum, is

known to infect multiple animal species and cause human fatalities in the U.S., Europe and

Asia. Although long known to infect ruminants, it is unclear why there are increasing

numbers of human infections. We analyzed the genome sequences of strains infecting

humans, animals and ticks from diverse geographic locations. Despite extensive variability

amongst these strains, those infecting humans had conserved genome structure including

the pfam01617 superfamily that encodes the major, neutralization-sensitive, surface

antigen. These data provide potential targets to identify human-infective strains and have

significance for understanding the selective pressures that lead to emergence of disease in

new species.

OPEN ACCESS

mailto:[email protected]





Pathogens 2013, 2 545

Keywords: anaplasmosis; tick-borne diseases; high-throughput sequencing; pfam01617;

msp2/p44; comparative genomics

1. Introduction

Human exposure to vectors carrying disease agents has been increased by climate and land-use

changes causing more contact between humans and domestic animals with wildlife reservoirs [1]. One

such recently emerging disease is tick-borne anaplasmosis that causes infections in multiple animal

species [2]. These include cattle, sheep, goats, horses, dogs, foxes, cats, rodents and most recently,

humans. Different strains of the causative organism, Anaplasma phagocytophilum, have different host

predilections and not all strains can infect all hosts. Small mammals are thought to be a reservoir,

infecting immature stages of ticks which act as bridge vectors transferring infection to humans and

domestic animals. Cases reported to the U.S. Centers for Disease Control and Prevention comprise a

flu-like febrile illness with some severe sequelae such as multiple organ failure, or severe acute

respiratory distress syndrome. In the U.S., case reports increased from 348 cases in 2000 to 1,761 cases

in 2010 [3]. The reported hospitalization rate is 36% [4]. Granulocytic anaplasmosis (GA) can be

treated with antibiotics, but the symptoms, such as headache, fever, and muscle aches are non-specific

and can be confused with other common diseases such as Lyme, transmitted by the same species of

tick. If left untreated GA can be severe [5], resulting in a case fatality rate in the U.S. of up to 3%

(CDC data for 2003). Increasingly, there are reports of infections transmitted by blood transfusions in

the U.S. and Europe [6,7]. Here we show that, in contrast to the extensive worldwide genomic

diversity of A. phagocytophilum strains, human-infective strains are a conserved subset. This has

implications for understanding the selective pressures that lead to emergence of disease in new species

and for control of this infection.

2. Results and Discussion

2.1. Comparative Genomics of Nine Strains of A. phagocytophilum

Underlying the ecological complexity and variable host tropism of A. phagocytophilum is a large

degree of genetic variability. European strains of A. phagocytophilum are highly pathogenic in sheep

and cattle, whereas North American strains do not cause disease in domestic ruminants. Some U.S.

strains, defined as Ap-variant 1 because of a 2 base pair difference in 16S rRNA, are infectious to deer

and other ruminants, but do not infect mice and are thought not to infect humans [8,9]. Highly unusual

for an obligate intracellular pathogen with a small, 1.5 Mb genome, A. phagocytophilum contains

approximately 100 “functional pseudogenes” of the major surface protein 2 gene (msp2/p44) that

codes for the major antigen on its surface [10]. These hypervariable pseudogene cassettes are

recombined into a single expression site which provides N- and C-terminal conserved sequences,

allowing the bacterium to serially express variable antigens and evade host immunity [11,12]. We

applied high-throughput genome sequencing to nine strains, derived from humans and different host

animal species from the U.S. and Europe. These strains are: two human origin strains from New York


and Minnesota, a rodent- and dog-origin strain from Minnesota, two Ap-variant 1 strains from

Minnesota, a horse-origin strain from California and two sheep-origin strains from Norway, for which

no in vitro cultures are available. In Figure 1 the complete genome sequence of a strain derived from

rodents in an area of high prevalence for human disease (Camp Ripley, MN, USA) is compared with

that of the human ApHZ strain (from the state of New York). The figure shows Blast comparisons of

the rodent and human strains, particularly the positions of members of the pfam01617 superfamily, to

which msp2/p44 belongs. Overall, despite widely separated geographic and species origins, the two

genomes are conserved and almost completely syntenic. The majority of the highly repetitive

msp2/p44 genes (visualized using partial opacity) are located in the third of the genome closest to the

origin of replication (top of figure, changing sign of GC skew), as reported previously [13]. However,

using high-stringency Blast comparisons (fifth circle from the outside) differences between the two

genomes were primarily localized to this region also and to msp2/p44 genes in particular, showing the

propensity of this gene family to undergo rapid diversification.

Figure 1. Genome synteny between two strains of A. phagocytophilum, ApHZ (human

origin, NY, USA) and ApJM (rodent origin, MN, USA). The reference strain is ApHZ

(GenBank CP000235). The two outermost circles show annotated ApHZ coding sequences,

the third circle shows the locations of members of the ApHZ pfam01617 superfamily, the

fourth and fifth circles are Blastn comparisons of the ApJM genome with ApHZ using 90%

and 99% identity cutoffs respectively, the sixth circle shows GC skew and the innermost

circle the numeric genome position. Comparisons were conducted using the CGView

server using partial opacity to visualize overlapping hits (darker bars in the green and blue

circles). The white bars on the fifth circle indicate no match.


This comparative genomics analysis was extended to all nine strains (Figure 2). Except for

Ap-variant 1 strains, the U.S. strains had high identities across their entire genomes, with the ApDog

strain most similar to human ApHZ by Blast analysis. The Norwegian sheep and U.S. Ap-variant 1

strains had the lowest identities with ApHZ in Blast comparisons, especially in members of the

pfam01617 superfamily.

Figure 2. Similarities and differences between nine A. phagocytophilum genomes.

Roche/454 reads from each genomic DNA were compared with the human ApHZ genome

as reference using Blastn and the CGView Comparison Tool. The first to ninth circles

compare, respectively, sequencing reads from ApHZ, ApDog, ApJM, ApHGE1, ApMRK,

ApCRT35, ApCRT38-1, ApNorV2, ApNorV1 genomic DNAs. The 10th circle shows the

locations of members of the ApHZ pfam01617 superfamily. Blast parameters used a query

size of 500 bp segments of the reference genome and a Blast expect value of 10−100

. Circles

are colored according to the percent identities of matches (black to light red, 100%–90%

identical, dark to light blue, 88%–82% identical, colorless, 0% identical).

The differences in this superfamily were examined more closely in order to determine their

msp2/p44 genomic repertoires and if differences were primarily located in the known pseudogene

hypervariable regions (Figure 3).


Figure 3. Gene variation in a region rich in p44/msp2 pseudogenes close to the origin of

replication in nine genomes of A. phagocytophilum. Roche/454 pyrosequencing reads from

each of the nine genomes are shown aligned with the annotated reference ApHZ genome

(bottom) between positions 1,450 and 1,462 kbp. The sequencing reads are derived from:

ApHZ, ApDog, ApHGE1, ApJM, ApMRK, ApCRT35, ApCRT38-1, ApNorV1, ApNorV2

(bottom to top panels respectively). The positions of six msp2/p44 pseudogenes are marked

by arrows at the top and shown in their respective reading frames at the bottom. The

hypervariable regions LAKT.. LAKT, present in five of the six pseudogenes, are indicated

in red. Gaps in coverage indicate no aligning reads over the central hypervariable regions

in some strains.

Similar to the rodent strain ApJM, the msp2/p44 repertoires of a U.S. human strain, ApHGE1 and

the ApDog strain from the same area of Minnesota were closely related and slightly different from the

New York human strain ApHZ (8/95 or 9/95 different msp2/p44 pseudogenes determined as <90%

identical, Table 1). In contrast, strains infecting horses or ruminants (Ap-variant 1 strains) in the U.S.

or Norway (representing “classical” strains long known to be pathogenic to sheep [14] shared fewer

msp2/p44 pseudogenes with human strains, with the two Norwegian sheep strains having almost

totally different repertoires from human strains. Differences between individual members of the

msp2/p44 repertoire were located in the known central hypervariable region between flanking

consensus amino acids LAKT.. LAKT (Figure 3).


Table 1. Genome structural relationships between Anaplasma strains.

Strain ApHZ ApHGE1 ApJM ApDog ApMRK ApCRT35 ApCRT38 ApNorV1 ApNorV2

Different msp2/p44

pseudogenes from ApHZ 0/95 8/95 8/95 9/95 49/95 71/95 75/95 92/95 89/95

ANIm 100 98.81 98.84 98.79 97.76 96.28 96.21 94.92 95.87

Tetra 1.000 0.999 0.999 0.999 0.998 0.996 0.996 0.995 0.996

Strain AmFL AmStM AcIs ApHZ

ANIm 100 98.86 88.89 77.82

Tetra 1.000 0.999 0.989 0.821

The top panel compares strains with ApHZ, the lower panel compares strains and species with AmFL. Abbreviations:

ANIm, Average % genome nucleotide identity using Mummer; Tetra, correlation coefficient of tetranucleotide signature

frequencies; AmFL, Anaplasma marginale Florida strain; AmStM, A. marginale St. Maries Idaho strain; AcIs,

Anaplasma centrale Israel strain; Ap, A. phagocytophilum strains.

Two additional quantitative measures of genome structural relationships compare the average

nucleotide identities or the tetranucleotide frequencies between two genomes. The latter method is

independent of alignment algorithms. The proposed threshold for prokaryotic species separation is 94%

for average nucleotide identity and 0.990 for the correlation coefficient of tetranucleotide signature

frequencies [15]. It is evident from Table 1 that the existing taxonomy of Anaplasma species meets these

definitions. However, it is apparent that by these measures also, the U.S. human, dog and rodent strains

are closely related (average nucleotide identity 98.79%–98.84% with ApHZ). The Norwegian ruminant

strains are again most distinct from ApHZ (average nucleotide identity 94.92%–95.87%), agreeing with

the similar divergence observed in their pfam01617 superfamily genes.

2.2. Comparison of the msp2/p44 Family among Strains of A. phagocytophilum

Although these data showed that two U.S. A. phagocytophilum strains infecting humans, as well as

a dog and rodent strain were similar to one another and different from strains infecting ruminants, we

wanted to analyze this more globally using a larger dataset. To do this, we took advantage of the fact

that conserved regions of msp2/p44 have been used as sensitive PCR diagnostic targets because of the

large copy number of the pfam01617 superfamily. More than 500 partial pseudogene sequences are

present in GenBank, from humans, ticks and animal strains derived from multiple regions throughout

the U.S., Europe and Asia. From these sequences it is possible to extract the hypervariable region of

msp2/p44 flanked by sequence encoding a consensus LAKT on either end, facilitating alignment [16].

Importantly, for this analysis it is necessary to recognize that multiple different hypervariable regions

exist in a single genome because of the ~100 distinct pseudogene cassettes present. Therefore, it is not

sufficient to simply analyze phylogenetic trees and apparent evolutionary relationships between all

msp2/p44 sequences. If one wishes to compare global repertoires with the genome-sequenced human

ApHZ strain, it is necessary to align each msp2/p44 sequence with all ~100 ApHZ strain genomic

pseudogene cassettes to find the best fit (maximum sequence identity). If strains are related to ApHZ,

one expects to find one or more ApHZ pseudogenes with high identity to other strain msp2/p44s. Our

analyses, therefore, employed a matrix where every available msp2/p44 polypeptide (661 total


including those encoded by ApHZ pseudogene cassettes) was aligned with every other, generating

218,791 alignments having a mean amino acid sequence identity of 48.2%. These percentage sequence

identities can be rapidly analyzed using a 661 (row) × 661 (column) spreadsheet. Surprisingly, despite

the differences in msp2/p44 repertoires observed previously, all 91 available msp2/p44 human-origin

sequences from widely dispersed U.S. locations (states of New York, Massachusetts, Wisconsin and

Minnesota) encoded polypeptides with a mean maximum percentage identity with ApHZ of 97.2%

(Table 2). Further, in analyzing the available msp2/p44 sequences it was notable that, world-wide,

other human-origin strains also achieved similar levels of sequence identity with ApHZ (e.g., 98.1%

mean maximum percentage amino acid identity of a dataset from Japan comprising 27 human-origin

msp2/p44 variants, no significant difference with ApHZ). In contrast, A. phagocytophilum strains from

Europe and Asia from non-human sources had mean maximum identities of <76% (significantly

different from ApHZ, Table 2).

Table 2. Comparison of msp2/p44 variants from different geographic locations with the

human ApHZ strain.

Country Source Number of msp2/p44

Variants Analyzed

Mean Maximum % a.a. Identity

with ApHZ (+/−Std.Dev.)

Significantly different

from U.S. Human *

U.S.A. Human 91 97.2 (7.0)

U.S.A. Dog 27 94.8 (9.8) No

U.S.A. Horse 29 94.2 (7.3) No

U.S.A. Bear 4 92.4 (4.1) N/A

U.S.A. Woodrat 88 78.4 (13.8) Yes

U.S.A. Ruminant, tick (Ap-variant 1) 34 86.5 (14.7) Yes

Norway Sheep 54 66.6 (6.4) Yes

Sweden Dog 19 64.3 (7.9) Yes

U.K. Goat 20 67.2 (8.2) Yes

U.K. Sheep 19 65.1 (6.7) Yes

Czech

Republic

Human, Roe deer, Perdix,

Ixodes ricinus 6 86.6 (14.5) N/A

[Human only] [2] [97.8][(2.0)] N/A

Japan Sika deer 17 39.3 (3.0) Yes

Japan Ixodes persulcatus 87 69.0 (6.9) Yes

Japan Ixodes ovatus 22 71.8 (15.5) Yes

Japan Haemaphysalis formosensis 9 75.6 (14) N/A

Japan Human 27 98.1 (7.2) No

China Human 2 100 (0) N/A

* Kruskal-Wallis One Way Analysis of Variance on Ranks (p ≤ 0.001) followed by Dunn’s method for Multiple

Comparisons versus a Control group (p ≤ 0.05). N/A: Not applied to sample sizes <10.

3. Experimental

3.1. Origin of A. phagocytophilum Strains

The origins of the A. phagocytophilum strains used in this study are as follows: ApHZ, human,

Westchester County, New York; HGE1, human, Minnesota; ApJM, meadow jumping mouse (Zapus


hudsonius), Camp Ripley, Minnesota; ApDog, Minnesota; ApCRT35, Camp Ripley tick (Ixodes

scapularis), Minnesota (defined previously as Ap-variant 1); ApCRT38-1, Camp Ripley tick (I.

scapularis), Minnesota (Ap-variant 1); ApMRK, horse, California; ApNorV1, sheep, Norway;

ApNorV2, sheep, Norway. A. phagocytophilum genomic DNA was prepared from in vitro cultured

organisms or directly from infected sheep (Norwegian strains), as described previously [17].

3.2. Ethics Statement

The experimental study in sheep was approved by the Norwegian Animal Research Authority.

3.3. Genome Sequencing and Bioinformatics

Genomic DNA was sequenced on the Roche/454 Genome Sequencer using non-paired and 3 kb

paired-end libraries, also as described [17]. Mean genome coverage with respect to ApHZ varied

between 31.3X and 72.1X. The ApJM sequence was finished using manual inspection for conflicts and

mismatched paired ends and PCR to fill gaps. All sequences were compared with ApHZ for regions of

identity using Blastn analysis in CGView [18,19]. Differences in the msp2/p44 repertoires between

strains were defined using a method previously validated using the pfam01617 superfamily and two

completely Sanger-sequenced genomes of Anaplasma marginale [20]. Briefly, this method uses

Mosaik to align individual reads and generate BAM format files to detect gaps in alignment (no

coverage) with respect to the annotated reference sequence. In A. marginale, this method detected all

different pseudogenes having <90% nucleotide identity with the reference genome. In homologous

comparisons between ApHZ Roche/454 reads and the ApHZ genome all msp2/p44 pseudogenes were

detected as present (Table 1). Comparisons of average nucleotide identities and the correlation

coefficients of tetranucleotide signature frequencies between genomes were conducted using Jspecies

software, as described [15].

3.4. Analysis of msp2/p44 Repertoires

To analyze global msp2/p44 repertoires, deposited msp2/p44 sequences were downloaded from

GenBank following Blast searches; several large datasets are also available from published

studies [16,21–25]. The sequences were each trimmed to that encoding the hypervariable region,

where possible using the consensus flanking LAKT residues, and the polypeptides were aligned (all

against all) with MATGAT [26]. This generates the alignments that can be inspected for accuracy, and

allows export of all percent identity values to a spreadsheet. This spreadsheet was analyzed in Excel

for the relatedness of the different datasets. The percent identity of each variant msp2/p44 sequence

with every ApHZ msp2/p44 polypeptide encoded by a pseudogene was reported and, from that, the

mean best match with ApHZ (maximum percent identity) determined for each dataset. As the

percentage identities were not normally distributed and the population variances were unequal,

significant differences between groups were determined by nonparametric methods (Table 2).


4. Conclusions

These data show that there is genome diversity worldwide within the A. phagocytophilum species

that extends close to some proposed guidelines for species discrimination. However, strains infecting

humans are a subset with more conserved genome structure than the species overall, and this includes

their repertoires of msp2/p44 genes. This subset of A. phagocytophilum strains does not differ

significantly from the U.S. ApHZ strain and is closely related to strains infecting U.S. domestic dogs.

The mean seroprevalence (using a conserved msp2/p44 peptide as antigen) in the U.S. among 479,640

dogs was 4.8% with prevalence >50% in some counties in the Northeast and Midwest, corresponding

to the location of the majority of human cases [27]. These data have significance in several areas. First,

msp2/p44 encodes a protein that induces neutralizing antibodies against homologous strains of

A. phagocytophilum [28,29]. The repertoire differences between strains reflect an evolving antigenic

system with adaptive pressures imposed by an array of different persistently infected hosts. In contrast,

humans are incidental dead-end hosts that do not impose significant pressure for change. The

similarities between human-origin strains in the U.S., Europe and Asia suggest that humans may not be

susceptible to many of the circulating wildlife strains. They may become susceptible when selection

pressures in small mammal reservoir hosts cause evolution of novel strains that allow invasion and survival

in humans. Rodents are important reservoirs for a multitude of human disease agents, vector-borne or not,

and are known to be infected with strains of A. phagocytophilum having different species tropisms [30].

Rodent control has historically been emphasized as a means to control human disease outbreaks. The

underlying evolutionary drivers remain to be identified but could reflect the long-standing association

between “men and mice”. Second, these data have practical significance for control of this emerging

infection. Despite the ubiquity of A. phagocytophilum strains and positive serology world-wide, it is

necessary to focus on the prevalence and transmission of a smaller, human-infective subset.

Differentiation of such strains from the high-prevalence background may be possible using genomic

PCR targets or selected msp2/p44 polypeptides for serology. In order to accomplish this goal it is

necessary to acquire genome sequences from multiple human-origin strains on different continents.

The technology now exists to do this.

Acknowledgments

We thank Anna M. Lundgren for technical support, Savita Shanker for high-throughput DNA

sequencing, David Allred and Dan Brown for reading the manuscript and to Dan Brown for suggesting

the use of Jspecies software. This investigation received support from grants RO1 GM081714

and GM081714-03S1.

Draft and complete sequences obtained in this project are being made available through the NCBI

Bioproject portal [31] as bioprojects PRJNA158483, 163167, 163169, 171710, 183838, 216999,

217002, 217033, and 217037.

Conflicts of Interest

The authors declare no conflict of interest.


References

1. Patz, J.A.; Olson, S.H.; Uejio, C.K.; Gibbs, H.K. Disease emergence from global climate and land

use change. Med. Clin. North Am. 2008, 92, 1473–1491.

2. Dumler, J.S.; Choi, K.S.; Garcia-Garcia, J.C.; Barat, N.S.; Scorpio, D.G.; Garyu, J.W.; Grab, D.J.;

Bakken, J.S. Human granulocytic anaplasmosis and Anaplasma phagocytophilum. Emerg. Infect.

Dis. 2005, 11, 1828–1834.

3. CDC. Statistics and Epidemiology. Annual Cases of Anaplasmosis in the United States; Available

online: www.cdc.gov/anaplasmosis/stats/ (accessed on 5 September 2013).

4. Dahlgren, F.S.; Mandel, E.J.; Krebs, J.W.; Massung, R.F.; McQuiston, J.H. Increasing incidence

of Ehrlichia chaffeensis and Anaplasma phagocytophilum in the United States, 2000–2007. Am. J.

Trop. Med. Hyg. 2011, 85, 124–131.

5. Zhang, L.; Wang, G.; Liu, Q.; Chen, C.; Li, J.; Long, B.; Yu, H.; Zhang, Z.; He, J.; Qu, Z.; et al.

Molecular analysis of Anaplasma phagocytophilum isolated from patients with febrile diseases of

unknown etiology in China. PLoS One 2013, 8, e57155.

6. Annen, K.; Friedman, K.; Eshoa, C.; Horowitz, M.; Gottschall, J.; Straus, T. Two cases of

transfusion-transmitted Anaplasma phagocytophilum. Am. J. Clin. Pathol. 2012, 137, 562–565.

7. Jereb, M.; Pecaver, B.; Tomazic, J.; Muzlovic, I.; Avsic-Zupanc, T.; Premru-Srsen, T.;

Levicnik-Stezinar, S.; Karner, P.; Strle, F. Severe human granulocytic anaplasmosis transmitted

by blood transfusion. Emerg. Infect. Dis. 2012, 18, 1354–1357.

8. Massung, R.F.; Priestley, R.A.; Miller, N.J.; Mather, T.N.; Levin, M.L. Inability of a variant strain

of Anaplasma phagocytophilum to infect mice. J. Infect. Dis. 2003, 188, 1757–1763.

9. Massung, R.F.; Mather, T.N.; Levin, M.L. Reservoir competency of goats for the Ap-variant 1

strain of Anaplasma phagocytophilum. Infect. Immun. 2006, 74, 1373–1375.

10. Dunning Hotopp, J.C.; Lin, M.; Madupu, R.; Crabtree, J.; Angiuoli, S.V.; Eisen, J.A.; Seshadri, R.;

Ren, Q.; Wu, M.; Utterback, T.R.; et al. Comparative genomics of emerging human ehrlichiosis

agents. PLoS Genet. 2006, 2, e21.

11. Barbet, A.F.; Meeus, P.F.; Bélanger, M.; Bowie, M.V.; Yi, J.; Lundgren, A.M.; Alleman, A.R.;

Wong, S.J.; Chu, F.K.; Munderloh, U.G.; et al. Expression of multiple outer membrane protein

sequence variants from a single genomic locus of Anaplasma phagocytophilum. Infect. Immun.

2003, 71, 1706–1718.

12. Lin, Q.; Rikihisa, Y. Establishment of cloned Anaplasma phagocytophilum and analysis of p44 gene

conversion within an infected horse and infected SCID mice. Infect. Immun. 2005, 73, 5106–5114.

13. Foley, J.E.; Nieto, N.C.; Barbet, A.F.; Foley, P. Antigen diversity in the parasitic bacterium

Anaplasma phagocytophilum arises from selectively-represented, spatially clustered functional

pseudogenes. PLoS One 2009, 4, e8265.

14. Stuen, S. Anaplasma phagocytophilum—The most widespread tick-borne infection in animals in

Europe. Vet. Res. Commun. 2007, 31, 79–84.

15. Richter, M.; Rosselló-Móra, R. Shifting the genomic gold standard for the prokaryotic species

definition. Proc. Natl. Acad. Sci. USA 2009, 106, 19126–19131.


16. Barbet, A.F.; Lundgren, A.M.; Alleman, A.R.; Stuen, S.; Bjöersdorff, A.; Brown, R.N.;

Drazenovich, N.L.; Foley, J.E. Structure of the expression site reveals global diversity in MSP2

(P44) variants in Anaplasma phagocytophilum. Infect. Immun. 2006, 74, 6429–6437.

17. Al-Khedery, B.; Lundgren, A.M.; Stuen, S.; Granquist, E.G.; Munderloh, U.G.; Nelson, C.M.;

Alleman, A.R.; Mahan, S.M.; Barbet, A.F. Structure of the type IV secretion system in different

strains of Anaplasma phagocytophilum. BMC Genomics 2012, 13, e678.

18. Grant, J.R.; Stothard, P. The CGView Server: A comparative genomics tool for circular genomes.

Nucleic Acids Res. 2008, 36, W181–W184.

19. Grant, J.R.; Stothard, P. Comparing thousands of circular genomes using the CGView

Comparison Tool. BMC Genomics 2012, 13, e202.

20. Dark, M.J.; Al-Khedery, B.; Barbet, A.F. Multistrain genome analysis identifies candidate vaccine

antigens of Anaplasma marginale. Vaccine 2011, 29, 4923–4932.

21. Casey, A.N.J.; Birtles, R.J.; Radford, A.D.; Bown, K.J.; French, N.P.; Woldehiwet, Z.;

Ogden, N.H. Groupings of highly similar major surface protein (p44)-encoding paralogues: A

potential index of genetic diversity amongst isolates of Anaplasma phagocytophilum.

Microbiology 2004, 150, 727–734.

22. Gaowa; Wuritu; Wu, D.; Yoshikawa, Y.; Ohashi, N.; Kawamori, F.; Sugiyama, K.; Ohtake, M.;

Ohashi, M.; Yamamoto, S.; et al. Detection and characterization of p44/msp2 transcript variants

of Anaplasma phagocytophilum from naturally infected ticks and wild deer in Japan. Jpn. J.

Infect. Dis. 2012, 65, 79–83.

23. Wuritu; Gaowa; Kawamori, F.; Aochi, M.; Masuda, T.; Ohashi, N. Characterization of p44/msp2

multigene family of Anaplasma phagocytophilum from two different tick species, Ixodes

persulcatus and Ixodes ovatus, in Japan. Jpn. J. Infect. Dis. 2009, 62, 142–145.

24. Ohashi, N.; Gaowa; Wuritu; Kawamori, F.; Wu, D.; Yoshikawa, Y.; Chiya, S.; Fukunaga, K.;

Funato, T.; Shiojiri, M.; et al. Human granulocytic anaplasmosis, Japan. Emerg. Infect. Dis. 2013,

19, 289–292.

25. Caspersen, K.; Park, J.H.; Patil, S.; Dumler, J.S. Genetic variability and stability of Anaplasma

phagocytophila msp2 (p44). Infect. Immun. 2002, 70, 1230–1234.

26. Campanella, J.J.; Bitincka, L.; Smalley, J. MatGAT: An application that generates

similarity/identity matrices using protein or DNA sequences. BMC Bioinformatics 2003, 4, e29.

27. Bowman, D.; Little, S.E.; Lorentzen, L.; Shields, J.; Sullivan, M.P.; Carlin, E.P. Prevalence and

geographic distribution of Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and

Anaplasma phagocytophilum in dogs in the United States: Results of a national clinic-based

serologic survey. Vet. Parasitol. 2009, 160, 138–148.

28. Kim, H.Y.; Rikihisa, Y. Characterization of monoclonal antibodies to the 44-kilodalton major

outer membrane protein of the human granulocytic ehrlichiosis agent. J. Clin. Microbiol. 1998,

36, 3278–3284.

29. Wang, X.; Kikuchi, T.; Rikihisa, Y. Two monoclonal antibodies with defined epitopes of P44

major surface proteins neutralize Anaplasma phagocytophilum by distinct mechanisms. Infect.

Immun. 2006, 74, 1873–1882.


30. Foley, J.E.; Nieto, N.C.; Massung, R.; Barbet, A.; Madigan, J.; Brown, R.N. Distinct ecologically

relevant strains of Anaplasma phagocytophilum. Emerg. Infect. Dis. 2009, 15, 842–843.

31. The NCBI Bioproject Portal. Available online: www.ncbi.nlm.nih.gov/bioproject (accessed on 5

September 2013).

© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article

distributed under the terms and conditions of the Creative Commons Attribution license

(http://creativecommons.org/licenses/by/3.0/).

an emerging tick-borne disease of humans is...

Documents